Effect of pH on the production of lipolyzed butter oil by a calf pregastric esterase immobilized in a hollow-fiber reactor: II. Multiresponse kinetics

The lipolysis of butter oil in a hollow-fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C and at pH values of 4.0, 5.0, 6.0, and 7.0. The concentrations of ten fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography (HPLC). The relative specificity of this esterase depended on pH. Three mathematical models derived from a generalized Michaelis-Menten mechanism were tested for their ability to describe the rates of release of individual specific fatty acids. Loss of enzyme activity was modeled using first order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The parameters of the model were also tested for dependence on pH. The model was successful in describing the rates of release of all ten fatty acid species for a range of space times and pH values.     

The Influence of Fatty Acid and Glycerol on the Kinetics of Fat Hydrolysis by Candida Rugosa Lipase in a Membrane Reactor

The kinetics of lipid-hydrolysis by Candida rugosa lipase was investigated in a membrane reactor and in an emulsion system. Two models were chosen to describe the kinetics of the enzyme:

(1) The hydrolysis of triglycerides to fatty acids was considered to be a chain reaction with the intermediary products di- and mono-glyceride; each step was assumed to be a reversible second-order reaction. The reaction rate constants were determined from batch experiments. The experimental results could be described with this model.

(2) For process optimization and control, a model based on the power law was developed. For this model, the rate of hydrolysis was measured as a function of fatty acid and glycerol concentrations. Relations for the initial rate and equilibrium ester fraction as a function of the glycerol concentration were determined. Further, the reaction rate could be described with the power-law model with a power of 1.75 in the hydrolyzable ester fraction for a wide range of glycerol concentrations. The model with power 1.75 gave much better results when compared to a similar first order model. Although simpler, the first order model can not be used. The power law model was applied in the simulation of a reactor composed of three modules. The fatty acid production rate was calculated for this reactor system as a function of the outgoing glycerol concentration at different conditions.     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

Kinetics of glycerol uptake by the perfused rat liver. Membrane transport, phosphorylation and effect on NAD redox level.

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by L-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K-m of the glycerol phosphorylation was 10 muM and the K-i of the L-glycerol 3-phosphate inhibition was 50 muM. The maximum activity (V) was 3.70 mumoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K-i was found to be somewhat lower in the intact organ. At low glycerol concentrations, a steep concentration gradient exists across the liver cell membrane. The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of L-glycerol 3-phosphate, not to the rate of glycerol uptake.     

Acetate formation after short-term ethanol administration in man

The effect of an acute oral load of 0.5 g ethanol/kg body weight was studied in a group of 10 healthy male and one of 10 healthy female individuals. The following parameters were measured in the blood between 0 and 7 h after the start of the experiment: ethanol, acetate, glucose, free fatty acids, free glycerol, lactate, pyruvate, 3-hydroxybutyrate, acetoacetate. While the elimination of ethanol followed zero-order kinetics between 2 and 5 h, a steady-state concentration of 0.4 to 0.6 mM acetate in the serum was observed during the same time interval. Concomitantly, a significant decrease of free fatty acid and free glycerol concentrations was observed.     

Kinetic analysis of the Ca2+-dependent, membrane-bound, macrophage phospholipase A2 and the effects of arachidonic acid

The kinetics of the Ca2+-dependent, alkaline pH optimum, membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line were studied using various phosphatidylcholine (PC) and phosphatidylethanolamine (PE) substrates. This enzyme exhibits "surface dilution kinetics" toward PC in Triton X-100 mixed micelles, and the "dual phospholipid model" was found to adequately describe its kinetic behavior. With substrate in the form of sonicated vesicles, the dual phospholipid model should give rise to Michaelis-Menten type kinetics. However, the hydrolysis of dipalmitoyl-PC, 1-palmitoyl-2-oleoyl-PC, and 1-stearoyl-2-arachidonoyl-PC vesicles exhibited two distinct activities. Below 10 microM, the data appeared to follow Michaelis-Menten behavior, while at higher concentrations, the data could best be fit to a Hill equation with a Hill coefficient of 2. These PCs had Vmax values for the low substrate concentration range of 0.2-0.6 nmol min-1 mg-1 and Km values of 1-2 microM. At the high substrate concentration range, the Vmax values were between 5 and 7 nmol min-1 mg-1. PC containing unsaturated fatty acids had an apparent Km, determined from the Hill equation, of about 15 microM, while the apparent Km of dipalmitoyl-PC was 0.6 microM. When 70% glycerol was included in the assays, a single Michaelis-Menten curve was obtained for both dipalmitoyl-PC and 1-stearoyl,2-arachidonoyl-PC. Possible explanations for these kinetic results include reconstitution of the membrane-bound phospholipase A2 in the phospholipid vesicle or the enzyme has tow distinct phospholipid binding function. The kinetics for both dipalmitoyl-PC and dipalmitoyl-PE hydrolysis in vesicles was very similar, indicating that the enzyme does not greatly prefer one of these head groups over the other. The enzyme also showed no preference for arachidonoyl containing phospholipid. Enzymatic activity toward PC containing saturated fatty acids was linear to about 15% hydrolysis while the hydrolysis of PC containing unsaturated fatty acids was linear to only about 5%. This loss of linearity was due to inhibition by released unsaturated fatty acids. Arachidonic acid was found to be a competitive inhibitor of dipalmitoyl PC hydrolysis with a K1 of 5 microM. This tight binding suggests a possible in vivo regulatory role for arachidonic acid. Three compounds of the arachidonic acid cascade, prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, showed no inhibition of enzymatic activity.     

Isolation and characterization of a murine serum esterase which hydrolyzes a tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate

An enzyme which catalyzes the following esterase reaction was isolated from mouse serum: 12-O-tetradecanoyl phorbol 13-acetate (TPA) + H2O----phorbol 13-acetate + tetradecanoic acid. The recovery was 0.18% of total serum protein and 820-fold purification was achieved. The enzyme is composed of a single polypeptide chain with sugar moiety; its molecular weight was estimated to be 77,000. Its sugar content is 15%, the isoelectric point was 4.3, and the alpha-helix content was 15.3% . The activity is stable between pH 5 and 9 under 40 degrees C; it is insensitive to 2-mercaptoethanol and is not dependent on divalent cations. The optimal pH is around 7.5. The apparent Km for TPA is 6.6 X 10(-7)M. The hydrolysis of [3H]TPA is inhibited by phorbol diesters and phorbol 12-myristate, but not by phorbol and phorbol 13-acetate. The activity is inhibited to some extent by phosphatidylcholine, cholesterol, and lanosterol, but not by free fatty acids, fatty acid esters of glycerol, cholesterol esters, or cholestanol. The enzyme hydrolyzes ester linkages, but not peptide linkages of synthetic substrates. Esterase inhibitors and serine-reactive reagents affect the activity. Although sera from rodents displayed strong activity, such activity was not detected in human serum. Unlike lipoprotein lipase, the serum enzyme activity was not enhanced by treatment of the animal with heparin. These characteristics and the amino acid composition do not agree with any of the reported characteristics of known serum enzymes with esterase activity.     

Some properties of hepatic glycerol kinase and their relation to the control of glycerol utilization

1. Glycerol kinase (EC 2.7.1.30) is shown to catalyse a non-equilibrium reaction in rat liver; and, as it is the first enzyme in the pathway metabolizing glycerol, its properties may be pertinent to the metabolic regulation of glycerol uptake and utilization by this tissue. 2. The properties of hepatic glycerol kinase were studied by using a radiochemical technique to measure the enzyme activity. When the concentration of ATP is low the activity of glycerol kinase is inhibited by high concentrations of glycerol; but when the concentration of ATP is high there is no inhibition and the double-reciprocal plot is linear, providing a K(m) for glycerol of 3.16x10(-6)m. Glycerol kinase is activated by high ATP concentrations provided that the concentration of the second substrate (glycerol) is high; at low concentrations of glycerol ATP does not activate the enzyme so that the double-reciprocal plot is linear, providing a K(m) for ATP of 5.8x10(-5)m. It is suggested that these kinetics may be explained by a model similar to that described by Ferdinand (1966) for phosphofructokinase. 3. Hepatic glycerol kinase is inhibited by ADP and AMP, and raising the Mg(2+) concentration increases the inhibition by these two compounds; this suggests that ADP-Mg(2+) and AMP-Mg(2+) complexes are the inhibitory species. The physiological significance of these inhibitions may be to prevent phosphorylation of glycerol when the hepatic ATP concentration is low. It is suggested that this inhibition may provide an approach to the problem of measurement of rates of lipolysis by glycerol release in tissues that contain glycerol kinase (e.g. liver, kidney, muscle, adipose tissue). 4. Hepatic glycerol kinase is inhibited by l-3-glycerophosphate competitively with respect to glycerol. The physiological significance of this inhibition may be that factors that change the intracellular concentration of l-3-glycerophosphate could change glycerol uptake by the tissue. Thus it is suggested that thyroxine treatment or feeding rats on a diet high in glycerol, which increase the activity of glycerophosphate oxidase in liver and kidney cortex respectively, lead to an increased glycerol uptake through a decrease in the concentration of glycerophosphate in these tissues. It is known that ethanol administration decreases glycerol uptake by liver, and this can be explained by the increased concentration of l-3-glycerophosphate causing inhibition of glycerol kinase.     

Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system

The kinetics of the enzymatic transesterification between a mixture of triglycerides (oils) and methanol for biodiesel production in a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane reversed micellar system, using recombinant cutinase from Fusarium solani pisi as a catalyst, was investigated. In order to describe the results that were obtained, a mechanistic scheme was proposed, based on the literature and on the experimental data. This scheme includes the following reaction steps: the formation of the active enzyme–substrate complex, the addition of an alcohol molecule to the complex followed by the separation of a molecule of the fatty acid alkyl ester and a glycerol moiety, and release of the active enzyme. Enzyme inhibition and deactivation effects due to methanol and glycerol were incorporated in the model. This kinetic model was fitted to the concentration profiles of the fatty acid methyl esters (the components of biodiesel), tri-, di- and monoglycerides, obtained for a 24 h transesterification reaction performed in a stirred batch reactor under different reaction conditions of enzyme and initial substrates concentration.     

Properties of triacylglycerol lipase in a mitochondrial fraction from baker's yeast (Saccharomyces cerevisiae).

A triacylglycerol lipase in a mitochondrial fraction isolated from yeast (Saccharomyces cerevisiae) has been characterized and the hydrolysis studied kinetically using an insoluble artificial triacylglycerol suspension. 1. The triacylglycerol was hydrolyzed almost completely to fatty acids and glycerol. The lipase activity was inhibited by potassium fluoride and the sodium salts of -chloride, -glycocholate and -pyrophosphate as well as by protamine sulfate but at concentrations much too high to indicate that the lipase is a non specific esterase or a lipoprotein lipase. Also parachloromercuribenzoate inhibited the lipase activity. Inhibitory effect of fatty acid was observed at concentrations above 1mM. This inhibition may provide a regulatory mechanism of the lipase in vivo. 2. On the day of isolation the lipase activity of intact mitochondria at pH 7.5 and 30 degrees C was 400 nmol free fatty acid -h-1 - mg-1 at a triacylglycerol concentration of 9.0 mM. Sonication of the mitochondria increased the activity 2-3 fold. Freezing of the mitochondria also activated the lipase and this activation was dependent upon the freezing method, the concentration of mitochondrial protein and the presence of bovine serum albumin. 3. The particulate nature of the assay system was illustrated by the observation that the apparent Km value of the lipase increased with the concentration of mitochondrial protein. For each protein concentration the lipase had two apparent Km values when the activity was assayed with intact mitochondria, but only one when assayed with submitochondrial particles. At the same protein concentration the Km value for the latter was identical with the "low affinity" Km for the lipase in intact mitochondria.     

Arachidonic acid-containing phosphatidylcholine species are increased in selected brain regions of a depressive animal model: Implications for pathophysiology

The Flinders Sensitive Line (FSL) rat is a genetic animal model of depression. Following recent findings that the brain fatty acid composition of FSL is characterised by increased arachidonic acid (AA), we used electrospray tandem mass spectrometry and ¹H-NMR to examine lipid species in different brain areas. Cholesterol and sphingolipids were increased in the hypothalamus of the FSL rats. Furthermore, arachidonic acid-containing phosphatidylcholine (AA-PC) species were elevated with PC16:0/20:4, PC18:1/20:4 and PC18:0/20:4 (p<0.003) increased in the hypothalamus and striatum. In contrast, there was a decrease in some docosahexaenoic acid (DHA)-containing species, specifically PC18:1/22:6 (p<0.003) in the striatum and PE18:1/22:6 (p<0.004) in the prefrontal cortex. Since no significant differences were observed in the erythrocyte fatty acid concentrations, dietary or environmental causes for these observations are unlikely. The increase in AA-PC species which in this animal model may be associated with altered neuropathy target esterase activity, an enzyme involved in membrane PC homeostasis, may contribute to the depressive phenotype of the FSL rats.     

The use of Tween 20 in a sensitive turbidimetric assay of lipolytic enzymes

A turbidimetric esterase assay was developed using a Tween 20 solution in the presence of CaCl2 and Lysobacter enzymogenes esterase (EC 3.1.1.1) as the enzyme source. The reaction was followed by measuring the increase in the optical density at 500 nm (OD500) due to the hydrolytic release of the fatty acids from Tween 20 and their precipitation as the calcium salts. Concentrations of 1.8% Tween and 3 mM CaCl2 were found to be optimal for the assay of 0.036 to 0.15 esterase units in a 4-mL reaction mixture over a 30-min period. The esterase reactions were linear with time at least up to 1.2 OD500 and the rate of increase in the OD500 was proportional to the enzyme concentration. Low initial reaction rates were seen with low esterase activity, presumably because of the limited solubility of the fatty acid - calcium salt in a 1.8% Tween solution. This turbidimetric method is much simpler and at least 36 times more sensitive than the titrimetric assay with Tween 20, and at least four times more sensitive than a spectrophotometric assay with p-nitrophenyl palmitate. This assay has been used to determine the activities of cell-associated and excreted esterases produced by Lysobacter enzymogenes and Pseudomonas aeruginosa, and of lipolytic enzymes from porcine liver, Chromobacterium viscosum, Candida cylindracea, and wheat germ.     

The molecular species of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from sn-glycerol 3-phosphate in rat lung microsomes

The species pattern of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from [14C]glycerol 3-phosphate was measured using a newly developed HPLC technique yielding 13 molecular species. A direct comparison of these species patterns presupposes determination of the lipolytic activity of lung microsomes. The lipolytic activity was quantitatively determined by measuring the changes of the endogenous concentration of diacylglycerol, triacylglycerol and free fatty acids. The species pattern of endogenous diacylglycerol measured in the time-course of lipolysis did not show any changes up to an incubation period of 20 min, suggesting that the lipolytic activity showed only a very low selectivity for individual substrate species. Diisopropylfluorophosphate (5 mumol/mg microsomal protein) strongly decreased the lipolytic activities as well as the microsomal phosphatidate phosphohydrolase activity, as measured by means of exogenous phosphatidic acid, and also the generation of phosphatidic acid from [14C]glycerol 3-phosphate. In lung microsomes, labeled phosphatidic acid and diacylglycerols were synthesized from the endogenous free fatty acids and sn-[14C]glycerol 3-phosphate, which had previously been added. By addition of CDPcholine to the prelabeled microsomes the synthesis of phosphatidylcholine was measured. After hydrolysis of phosphatidic acid and phosphatidylcholine with cytoplasmatic phosphatidate phosphohydrolase or phospholipase C, respectively, the de novo synthesized species patterns of these two lipids and of the diacylglycerol were determined. Comparison of the species pattern of de novo synthesized phosphatidic acid with that of diacylglycerol largely showed the same distribution of radioactivity among the individual species, except that the relative proportion of label was higher in the 16:0/16:0 and 16:0/18:0 species of phosphatidic acid and lower in the 16:0/20:4 and 18:0/20:4 species than in the corresponding species of diacylglycerol. The species pattern of de novo-synthesized diacylglycerol showed no differences from that of the phosphatidylcholine synthesized from it. From this result we concluded that the cholinephosphotransferase of lung microsomes is nonselective for individual species of the diacylglycerol substrate. The 16:0/18:1 and 16:0/18:2 species of phosphatidic acid, diacylglycerol and phosphatidylcholine showed a higher synthesis rate than their 18:0 counterparts, whereas the 16:0 or 18:0 analogues of species containing 20:4 and 22:6 fatty acids showed nearly the same synthesis rates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Factors affecting the reassociation and reactivation of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The pigeon liver fatty acid synthetase complex (14 S) is dissociated in low ionic strength buffer containing dithiothreitol to form a half-molecular weight subunits (9 S) which are completely inactive for the synthesis of saturated fatty acids. The dithiothreitol-protected (reduced) subunits are rapidly reassociated and reactivated to form the active enzyme complex, not only by an increase in salt concentration but also by micromolar concentrations of NADP+ or NADPH. Increases in KCl or NADPH concentration result in an increase in the extent of reactivation (equilibrium) with no change in the over-all rate of the reaction or the half-life ofreactivation of the enzyme. The extent (equilibrium) of reactivation of the enzyme is the same in 0.2 M potassium phosphate buffer, pH 7.0; 0.2 M KCl in 5 mM Tris-35 mM glycine buffer, PH 8.3; and 50 muM NADP+ or NADPH in the Tris-glycine buffer. The extent and rate of reactivation of the enzyme is dependent not only on ionic strength and NADPH concentration, but also on pH and temperature. Reactivation with 0.2 M KCl is optimal between pH 7.3 and 8.5. At higher and lower pH values the rate and extent of reactivation are lowered. The rate and extent of reactivation are also decreased as the temperature is lowered below 10 degrees. At 0 degrees there is little reactivation of enzyme activity. However, in the presence of 0.2 M KCl containing 15 to 40% glycerol at 0 degrees, reactivation of the enzyme is about 50% complete. The rate of reactivation of enzyme in the presence of KCl or NADPH conforms to first order kinetics. This result suggests that the subunits first combine to form an inactive complex which is subsequently transformed to an enzymatically active complex. Evidence for the presence of inactive complex was obtained in experiments carried out in 0.2 M KCl at pH 6.0, and in 0.2 M KCl at pH 8.3, at both 6 and 3 degrees. Under these conditions the amount of complex observed upon ultracentrifugation was greater than expected from determinations of enzyme activity. The above findings suggest that ionic and hydrophobic interactions, and possibly the water structure surrounding the interacting sites, are of prime importance in reassociation and reactivation of enzyme. In addition, NADP+ and NADPH have very specific effects in bringing about reassociation and in maintaining the structural integrity of the multienzyme complex.     

Production,isolation and characterization of a sterol esterase from Ophiostoma piceae

We studied extracellular sterol esterase production by the ascomycete Ophiostoma piceae in liquid culture. Esterase activity was found in low levels in glucose medium but it was strongly induced by olive oil. An esterase was purified from the 0.5% olive oil-supplemented cultures using ultrafiltration followed by a single chromatographic step on a hydrophobic interaction column. The enzyme was a glycoprotein with 8% N-linked carbohydrate content, a molecular mass by SDS/PAGE around 56.5 kDa and an isoelectric point of 3.3. Its N-terminal sequence was TTVNVKYPEGEVV. Substrate specificity studies showed that the O. piceae esterase hydrolyzes p-nitrophenol esters, tributyrin, triolein and different cholesterol esters. Both affinity (Km) and catalytic constant (k(cat)) were positively affected by the length of the fatty acid esterifying glycerol and cholesterol. The presence of double bonds in the acyl chain increased the enzyme efficiency, although it affected the k(cat) values rather than the Km on the cholesterol esters. The O. piceae enzyme showed no interfacial activation. This enzyme could have biotechnological applications in paper manufacturing since it efficiently hydrolyzes both triglycerides and sterol esters, which form pitch deposits during manufacturing of softwood and hardwood paper pulps, respectively.     

A cold-adapted esterase from psychrotrophic <Emphasis Type="Italic">Pseudoalteromas</Emphasis> sp. strain 643A

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli TOP10F', and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20-50% of maximum activity at 0-20 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2-mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions.     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Modulation of lipid-synthesizing enzymes by insulin in differentiated ob17 adipose-like cells.

ob17 cells convert into adipose-like cells when maintained in the presence of physiological concentrations of insulin and tri-iodothyronine. After this conversion, insulin removal from differentiated ob17 cells gives within 24-48 h a large decrease in fatty acid synthetase, glycerol 3-phosphate dehydrogenase and acid:CoA ligase activities, as well as in the rate of fatty acid synthesis determined by [14C]acetate incorporation into lipids. All parameters are restored by insulin addition to initial values within 24-48 h. Dose-response curves of insulin on the restoration of glycerol 3-phosphate dehydrogenase activity and of fatty acid synthesis give half-maximally effective concentrations close to 1 nM, in agreement with the affinity for insulin of the insulin receptors previously characterized in these cells. Immunotitration experiments indicate that the changes in the specific activity of fatty acid synthetase are due to parallel changes in the cellular enzyme content. Therefore the ob17 cell line should be a useful model to study the long-term effects of insulin on the modulation of lipid synthesis in adipose cells.     

Response surface modeling of 1-stearoyl-3(2)-oleoyl glycerol production in a pilot packed-bed immobilized Rhizomucor miehei lipase reactor

A dual response approach using diacylglycerol (DAG) and triacylglycerol (TAG) as responses for optimization of 1-stearoyl-3(2)-oleoyl glycerol-enriched DAG synthesis using response surface methodology (RSM) was investigated. Four variables from a lipase-catalyzed esterification reaction were optimized using a central composite rotatable design. The following optimized conditions yielded 51 wt.% DAG and 22 wt.% TAG: reaction temperature of 55 °C, enzyme dosage of 9.5 wt.%, fatty acid/glycerol molar ratio of 2.1 and reaction time of 3 h. Results were repeatable at 10 kg production scale in a pilot packed-bed enzyme reactor. No significant losses in enzyme activity or changes in fatty acid selectivity on DAG synthesis were observed during the five pilot productions. Lipozyme RM IM showed selectivity towards the production of stearic acid enriched DAG. The purity of DAG oil after purification was 90 wt.%.